Recombinant Viral Genomes and Related Compositions and Methods

Abstract

Provided are recombinant viral genomes. The viral genomes find use in a variety of contexts including for the production of viruses effective in inducing death of cells exhibiting a signal specific to a disease or disorder in a subject, where the viral life cycle is dependent upon the signal specific to the disease or disorder. According to some embodiments, the viral genomes comprise one or more transcription units comprising a sequence encoding a first fusion protein comprising a protease and a first binding domain that binds to a first target, and a sequence encoding a second fusion protein comprising a membrane-targeting signal, a second binding domain that binds to a second target, a cleavable substrate for the protease, and either a transcriptional effector protein or viral protein encoded by a viral gene deleted in the viral genome. Also provided are related cells, virions, pharmaceutical compositions and methods of use.

What is claimed is: 
     
         1 . A recombinant viral genome comprising:
 (a) a first transcription unit comprising:
 (i) a first sequence encoding a first fusion protein comprising a protease and a first binding domain that binds to a first target, and 
 (ii) a second sequence encoding a second fusion protein comprising, in N-terminal to C-terminal order or in C-terminal to N-terminal order, a membrane-targeting signal, a second binding domain that binds to a second target, a substrate cleavable by the protease, and a transcriptional effector protein; and 
   (b) a second transcription unit comprising a third sequence encoding one or more viral replication proteins, wherein the second transcription unit is operably linked to a promoter activatable by the transcriptional effector protein.   
     
     
         2 . The recombinant viral genome of  claim 1 , wherein the recombinant viral genome comprises a modified genome of a virus. 
     
     
         3 . The recombinant viral genome of  claim 2 , wherein the virus is an oncolytic virus. 
     
     
         4 . The recombinant viral genome of any one of  claims 1 to 3 , wherein the promoter is not activatable by the transcriptional effector protein until the transcriptional effector protein is cleaved from the second fusion protein. 
     
     
         5 . The recombinant viral genome of any one of  claims 1 to 3 , wherein the promoter is activatable to a lesser extent by the second fusion protein compared to the transcriptional effector protein when cleaved from the second fusion protein. 
     
     
         6 . The recombinant viral genome of any one of  claims 1 to 5 , wherein the transcriptional effector protein comprises a transcription factor. 
     
     
         7 . The recombinant viral genome of any one of  claims 2 to 6 , wherein the one or more viral replication proteins encoded by the second transcription unit is required for replication of the virus in cells or efficient replication of the virus in cells. 
     
     
         8 . The recombinant viral genome of any one of  claims 2 to 7 , wherein replication of the virus in cells is dependent on a signal specific to cancer cells or a signal in cancer cells that is higher than the signal in non-cancer cells. 
     
     
         9 . The recombinant viral genome of any one of  claims 2 to 8 , wherein replication of the virus in cells is not dependent on the presence of an endogenous transcription factor. 
     
     
         10 . The recombinant viral genome of  claim 9 , wherein the endogenous transcription factor is an endogenous cancer-selective transcription factor. 
     
     
         11 . The recombinant viral genome of any one of  claims 1 to 10 , wherein the recombinant viral genome further comprises a third transcription unit encoding an immunostimulatory molecule, optionally wherein the immunostimulatory molecule is granulocyte/macrophage colony stimulating factor (GM-CSF). 
     
     
         12 . The recombinant viral genome of any one of  claims 2 to 11 , wherein the virus is a double-stranded DNA virus. 
     
     
         13 . The recombinant viral genome of  claim 12 , wherein the double-stranded DNA virus is an adenovirus. 
     
     
         14 . The recombinant viral genome of  claim 13 , wherein the one or more viral replication proteins comprise one or more E1 gene products. 
     
     
         15 . The recombinant viral genome of any one of  claims 1 to 14 , wherein the transcriptional effector protein comprises a GAL4-VP16 transcription factor. 
     
     
         16 . The recombinant viral genome of  claim 15 , wherein the promoter activatable by the transcriptional effector protein is a GAL4 UAS promoter. 
     
     
         17 . A recombinant viral genome, comprising:
 (a) a deficiency in at least one native viral gene; and   (b) one or more transcription units comprising:
 (i) a sequence encoding a first fusion protein comprising a protease and a first binding domain that binds to a first target, and 
 (ii) a sequence encoding a second fusion protein comprising, in N-terminal to C-terminal order or in C-terminal to N-terminal order, a membrane-targeting signal, a second binding domain that binds to a second target, a substrate cleavable by the protease, and the viral protein encoded by the deleted gene. 
   
     
     
         18 . The recombinant viral genome of  claim 17 , wherein the recombinant viral genome comprises a modified genome of a virus. 
     
     
         19 . The recombinant viral genome of  claim 18 , wherein the virus is a wild-type virus. 
     
     
         20 . The recombinant viral genome of  claim 18 , wherein the virus is an engineered virus. 
     
     
         21 . The recombinant viral genome of  claim 18 , wherein the virus is an RNA virus. 
     
     
         22 . The recombinant viral genome of  claim 19 , wherein the RNA virus is a negative-strand RNA virus. 
     
     
         23 . The recombinant viral genome of  claim 22 , wherein the negative-strand RNA virus is a mononegavirus. 
     
     
         24 . The recombinant viral genome of  claim 23 , wherein the mononegavirus is a rhabdovirus. 
     
     
         25 . The recombinant viral genome of  claim 24 , wherein the rhabdovirus is a vesicular stomatitis virus (VSV). 
     
     
         26 . The recombinant viral genome of  claim 25 , wherein the viral protein encoded by the deleted gene is VSV phosphoprotein (VSVP). 
     
     
         27 . The recombinant viral genome of any one of  claims 1 to 26 , wherein the first and second targets are present on the same protein. 
     
     
         28 . The recombinant viral genome of  claim 27 , wherein the first and second targets are different epitopes of the same protein. 
     
     
         29 . The recombinant viral genome of any one of  claims 1 to 26 , wherein the first and second targets are present on different proteins. 
     
     
         30 . The recombinant viral genome of  claim 29 , wherein the different proteins form a complex with each other. 
     
     
         31 . The recombinant viral genome of  claim 30 , wherein the complex is present at a higher level in cancer cells than non-cancer cells. 
     
     
         32 . The recombinant viral genome of any one of  claims 1 to 28 , wherein the first and second targets are present on one or more proteins that are specifically expressed by cancer cells. 
     
     
         33 . The recombinant viral genome of any one of  claims 1 to 28 , wherein the first and second targets are present on one or more proteins that are expressed at a higher level in cancer cells compared to non-cancer cells. 
     
     
         34 . The recombinant viral genome of any one of  claim 1 to 28, 32, or 33 , wherein the first binding domain is a phosphotyrosine binding domain. 
     
     
         35 . The recombinant viral genome of any one of  claim 1 to 28, 32, or 33 , wherein the second binding domain is a phosphotyrosine binding domain. 
     
     
         36 . The recombinant viral genome of any one of  claim 1 to 28, 32, or 33 , wherein the first binding domain is a phosphotyrosine binding domain and the second binding domain is a phosphotyrosine binding domain. 
     
     
         37 . The recombinant viral genome of any one of  claims 1 to 36 , wherein the first and second targets are in proximity to each other such that the protease cleaves the cleavable substrate when the first and second binding domains are bound to their respective targets. 
     
     
         38 . The recombinant viral genome of  claim 27 , wherein the same protein is a receptor or a transmembrane protein. 
     
     
         39 . The recombinant viral genome of  claim 27 , wherein the same protein is hyperactive or signals constitutively. 
     
     
         40 . The recombinant viral genome of  claim 27 , wherein the same protein is a mutant protein. 
     
     
         41 . The recombinant viral genome of  claim 27 , wherein the same protein is human EGF receptor 2 (HER2). 
     
     
         42 . The recombinant viral genome of  claim 27 , wherein the same protein is epidermal growth factor receptor (EGFR). 
     
     
         43 . The recombinant viral genome of any one of  claims 1 to 42 , wherein the first binding domain of the first fusion protein and the second binding domain of the second fusion protein is independently selected from a Src homology 2 (SH2) domain and a Shc domain. 
     
     
         44 . The recombinant viral genome of any one of  claims 1 to 43 , wherein the protease is a viral protease. 
     
     
         45 . The recombinant viral genome of  claim 44 , wherein the viral protease is a hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease. 
     
     
         46 . The recombinant viral genome of any one of  claims 1 to 45 , wherein the membrane-targeting signal comprises a transmembrane domain. 
     
     
         47 . The recombinant viral genome of  claim 46 , wherein the membrane-targeting signal comprises a membrane-tethering domain. 
     
     
         48 . The recombinant viral genome of  claim 47 , wherein the membrane-tethering domain comprises a post-translational modification. 
     
     
         49 . The recombinant viral genome of  claim 48 , wherein the post-translational modification comprises palmitoylation, myristoylation, prenylation, a glycosylphosphatidylinositol (GPI) anchor, or any combination thereof. 
     
     
         50 . The recombinant viral genome of any one of  claims 1 to 49 , wherein the first fusion protein, the second fusion protein, or both, comprise a protein domain that emits a detectable signal. 
     
     
         51 . A cell comprising the recombinant viral genome of any one of  claims 1 to 50 . 
     
     
         52 . The cell of  claim 51 , wherein the cell is a mammalian cell. 
     
     
         53 . The cell of  claim 51 or claim 52 , wherein the cell is that of a viral packaging cell line. 
     
     
         54 . A virion produced by the cell of  claim 53 . 
     
     
         55 . A virion comprising the recombinant viral genome of any one of  claims 1 to 50 . 
     
     
         56 . A pharmaceutical composition comprising a plurality of virions of  claim 55 . 
     
     
         57 . A method of treating a disease or condition in a subject in need thereof comprising administering the pharmaceutical composition of  claim 56  to the subject. 
     
     
         58 . The method of  claim 57 , wherein the pharmaceutical composition comprises the virions in an amount effective to induce death of cells exhibiting a signal specific to the disease or disorder in the subject. 
     
     
         59 . The method of  claim 57 , wherein the pharmaceutical composition comprises the virions in an amount effective to induce death of cells exhibiting a signal that is higher than the signal in non-diseased cells. 
     
     
         60 . The method of  claim 57 , wherein the disease or condition is a cancer. 
     
     
         61 . The method of  claim 57 , wherein the disease or condition is characterized by hyperactive ErbB signaling. 
     
     
         62 . The method according to  claim 61 , wherein the disease or condition characterized by hyperactive ErbB signaling is a disorder characterized by hyperactive epidermal growth factor receptor (EGFR) signaling. 
     
     
         63 . The method according to  claim 61 , wherein the disease or condition characterized by hyperactive ErbB signaling is a disease or condition characterized by hyperactive human EGF receptor 2 (HER2) signaling. 
     
     
         64 . The method according to  claim 63 , wherein the disease or condition characterized by hyperactive ErbB signaling is a cell proliferative disease or condition. 
     
     
         65 . The method according to  claim 64 , wherein the cell proliferative disease or condition is a cancer. 
     
     
         66 . The method according to  claim 65 , wherein the cancer is selected from breast cancer, ovarian cancer, and non-small cell lung cancer. 
     
     
         67 . A method of producing a conditionally replicating or nonreplicating mononegavirus, the method comprising:
 (a) infecting a first cell population with a virus expressing a DNA sequence-specific RNA polymerase, wherein the first cell population comprises:
 (i) one or more plasmids containing one or more genes encoding a nucleocapsid, a phosphoprotein, and a large protein of a mononegavirus wherein the one or more genes are linked to a DNA sequence recognized by the RNA polymerase, and 
 (ii) a plasmid comprising a genome of the mononegavirus linked to the DNA sequence recognized by the DNA sequence-specific RNA polymerase; and 
   (b) culturing a second cell population with the first cell population and/or a culture medium thereof, wherein the second cell population expresses a nucleocapsid, a phosphoprotein, and a large protein, under one or more conditions in which the second cell population produces the mononegavirus.   
     
     
         68 . The method according to  claim 67 , wherein the DNA sequence-specific RNA polymerase is a bacteriophage T7 RNA polymerase. 
     
     
         69 . The method according to  claim 67 , wherein the mononegavirus is a rhabdovirus. 
     
     
         70 . The method according to  claim 69 , wherein the rhabdovirus is a VSV. 
     
     
         71 . The method according to any one of  claims 67 to 70 , wherein the first cell population comprises HEK293T cells. 
     
     
         72 . The method according to any one of  claims 67 to 71 , wherein the second cell population comprises BHK21 cells. 
     
     
         73 . The method according to any one of  claims 67 to 72 , wherein the second cell population comprises Vero cells. 
     
     
         74 . The method according to any one of  claims 67 to 73 , wherein the nucleocapsid, the phosphoprotein, or the large protein, or a combination thereof, are conditionally functional. 
     
     
         75 . The method according to any one of  claims 67 to 73 , wherein the nucleocapsid, the phosphoprotein, or the large protein, or a combination thereof, are nonfunctional.